1. Field of the Disclosure
The disclosure relates in general to a catalyst/enzyme structure and a fabricating method thereof, and more particularly to a homogeneously-structured catalyst/enzyme composite structure, a fabricating method and an application of the same. The method could be applied in a wide application such as an organic molecule sensor or a bioreceptor, such as an immobilization for a substrate of the mini sensor, etc.
2. Description of the Related Art
FIG. 1 illustrates a schematic diagram of a basic structure of one kind of a biosensor. The basic structure of the biosensor comprises a biological recognition element (bioreceptor) 11, a signal transducer 12 and a signal processor 13. During a theoretical sensing process of the biosensor, the variance quantity of the physical or chemical variance of the biological recognition element 11, having biochemical specificity, resulted from the binding or reacting with a test compound 15 would transform into a significant electronic signal through the signal transducer 12. The signal could be amplified and recorded by the signal processor 13 for convenience of subsequent qualitative and quantitative analysis.
Biological materials used as sensing elements for the biosensor usually comprise the following kinds: (a) enzyme, (b) antibody and antigen, (c) nucleic acid, (d) receptor, and (e) cell organelle. The biosensors fabricated by using different biological sensing materials have specific advantages and disadvantages. Among the many kinds of the biological sensing materials, enzymes are the most-early and most-frequently used. Generally speaking, the enzyme has characteristics of specificity (i.e. one kind of the enzyme can only catalyze one specific matrix), repeatability, heat susceptivity, acid-base susceptivity, etc. The catalytic behavior of binding with a test compound of an enzyme can be applied in a biosensor.
As universal economic development is improved, life and diet of human being are changed substantially. Globally, the main causes of death have been shifted from acute infections to chronic diseases. Among many chronic diseases, diabetes mellitus (often simply referred to as diabetes) is by far the most common chronic disease affecting great population. Statistically, diabetes takes the fourth place of ten major causes of death, published by National Health Administration of Taiwan in 2007. Although medical technology has made progress, diabetes usually cannot be cured completely. Diabetes without proper treatments and being controlled can cause many complications. This contributes significant burdens on individuals, families, societies and countries. For example, it not only consumes societal medical resources but also affect qualities of lives on a patient and families of that. A biosensor having an excellent performance can be used for properly monitoring and tracking a blood glucose concentration of a person for preventing and easing a complication. An intention of an early discovery and an early remedy can be also achieved. A mini sensor is a portable apparatus, which is easy to carry and measures a concentration of a test compound instantly.
There are a variety of technical combinations or methods could be adopted to make a completed biosensor for monitoring diabetes. In other words, a sensing device for analyzing glucose can be assembled by using many kinds of biochemical elements collocated with proper signal transducers. In many researches, an electrochemical glucose biosensor having glucose oxidases fixed therein is used for investigating analysis of the glucose. Therefore, glucose oxidases (GOD) are the most-frequently-used enzymes for these researches.
A glucose oxidase is one kind of compound comprising two similar glycoproteins of quadratic elements. The glycoproteins of quadratic elements are connected by a disulfide group. Each subunit comprises a complementation group of flavin adenine dinucleotide (FAD) having a following molecular structure:

In an existence of an electron substrate, the GOD could catalyze a β-D-glucose to become a D-glucono-δ-lactone. In addition, the FAD on the GOD would be reduced to form a FADH2, as shown as an equation (1-1):β-D-Glucose+GOD(FAD)→GOD(FADH2)+D-glucono-δ-latone  (1-1)
The D-glucono-δ-lactone would further react with the water in the solution to form a gluconic acid, as shown as a equation (1-2):D-glucono-δ-lactone+H2O→gluconic Acid  (1-2)
The equation (1-1) and the equation (1-2) would be combined to obtain an equation (1-3):β-D-Glucose+GOD(FAD)→GOD(FADH2)+gluconic Acid  (1-3)
The oxygen in the solution could be an electron acceptor of the GOD(FADH2). Therefore, the electron of the GOD(FADH2) would transfer to the oxygen and reduce the oxygen to become a hydrogen preoxide. In addition, the GOD(FADH2) is oxidized to become a GOD(FAD) shown as a equation (1-4):GOD(FADH2)+O2→GOD(FAD)+H2O2  (1-4)
The equation (1-4) and the equation (1-3) could be combined to obtain a equation (1-5):β-D-Glucose+O2→gluconic acid+H2O2  (1-5)
The biosensor as shown in FIG. 1 has advantages of easily performing an analysis treatment and an output display by transforming a biochemical signal into an electronic signal. The biosensor can be assembled as different types as being applied for different electronic devices. In FIG. 1, the signal transducer 12 can transform the physical or chemical variance quantity into an electronic signal that could be measured. In addition, in a proper condition, the strength of the electronic signal is also was proportioned to a concentration of a specific compound. Three types of the signal transducers 12 of different structures and energy conversion mechanisms are: (1) electrochemical biosensor, (2) optical biosensor, and (3) mass biosensor. The signal transducers 12 of different types have respective advantages and disadvantages.
The electrochemical biosensor uses an electrochemical measuring method using a potential, an electric current or a signal transducer, and a bioreceptor having a modified electrode used for carrying out a catalysis reaction of a test compound in a test sample with a fixed biological molecule for generating a product. As the product reacts with a catalyst on the surface of the electrode by an electrochemical oxidation-reduction reaction, the concentration of the test compound can be obtained by an indirect determination method using an output electric current or voltage, or a variance quantity of electric conductivity due to the reaction. During a process of sensing glucose of a conventional glucose receptor, for example, a glucose oxidase fixed on a surface of an electrode would react with a glucose in a test solution by a catalysis reaction to form hydrogen peroxide. Hydrogen peroxide would diffuse onto the surface of the electrode to react with the catalyst material by an electrochemical oxidation-reduction reaction to generate an electric current signal. Therefore, the electrochemical bioreceptor has the bioreceptor layer having specificity for the test compound. Moreover, the electrochemical bioreceptor also has the advantage of an electrochemical transducer. Expensive and complicated instruments are not necessary. The signal response time is very short. The linear detecting range is extensive. In addition, the operating process is simple and easy. Therefore, the convenience of the electrochemical biosensor is very helpful for future popularization for clinical application. Three types of the electrochemical transducers according to properties of electronic signals of which are a potentiometric type, an amperometric type, and a conductometric type. The amperometric type electrochemical transducer is used most frequently.
In a conventional method for fabricating a mini sensor, a catalyst is fixed on a working electrode of the mini sensor by a screen printing method. After the mini sensor is fabricated, an enzyme is fixed on the surface of which by an enzyme immobilization method. FIG. 2 illustrates a schematic diagram of a layer-shape structure of a conventional mini sensor. The disadvantage of the mini sensor fabricated by the conventional method comes from a structure of a layered stack of the catalyst/enzyme on the surface of the working electrode comprising a substrate 21 and a silver wire 22. The inner layer and the outer layer of the layered stack of the catalyst/enzyme are a catalyst layer 24 and an enzyme layer 26, respectively. For example, the enzyme layer 26 of the glucose oxidase, formed by the conventional immobilization method, on the surface of the electrode has a thickness of about 60 μm-80 μm. The enzyme layer 26 of a thickness of the layered structure as shown in FIG. 2 would be an obstructer obstructing the diffusion of hydrogen peroxide into the catalyst layer 24. In addition, the obstructer layer would result in a mass transfer resistance for the diffusion of hydrogen peroxide generated through the glucose oxidase on the surface into the surface of the electrode. Therefore, the sensing ability of the mini sensor is reduced. Especially, the sensitivity of the receptor for the glucose of high concentration is reduced.
Moreover, the conventional method for fabricating the mini sensor needs complicated steps. In addition, the catalyst and the enzyme are the expensive materials. However, the conventional method for fabricating the mini sensor having a desired sensing ability often requires excess of the catalyst and the enzyme to make. Therefore, the production cost of the mini sensor is very high.